Abstract
Previously, we showed that B-cell receptor associated protein 31 (BAP31), an endoplasmic reticulum (ER) membrane chaperone, is also expressed on the cell surface by two monoclonal antibodies (MAbs) 297-D4 and 144-A8. Both MAbs recognize the same linear epitope on the C-terminal domain of BAP31, although they were independently established. Here, flow cytometric analysis showed that 144-A8 had additional binding properties to some cells, as compared to 297-D4. Quantitative antigen binding assays also showed that 144-A8 had higher antigen binding capacity than 297-D4. Affinity measurement revealed that 144-A8 had 1.54-fold higher binding affinity than 297-D4. Analysis of the heavy- and light-chain variable region sequences of two MAbs revealed that both MAbs belonged to the same heavy chain (Igh-V3660 VH3) and light chain subgroup (IGKV21) with just two amino acid differences in each framework region, indicating that both MAbs arise from the same germline origin. Seven amino acid differences were found between the complementarity determining regions (CDRs) of the two MAbs. Molecular modeling of the epitope-paratope complexes revealed that the epitope appeared to reside in closer proximity to the CDRs of 144-A8 than to those of 297-D4 with the stronger hydrogen bond interactions with the former than the latter. More interestingly, an additional hydrophobic interaction appeared to be established between the leucine residue of epitope and the paratope of 144-A8, due to the substitution of H-Tyr101 for H-Phe101 in 144-A8. Thus, the different binding specificity and affinity of 144-A8 appeared to be due to the different hydrogen bonds and hydrophobic interaction induced by the alterations of amino acids in CDRs of 144-A8. The results provide molecular insights into how the binding specificities and affinities of antibodies evolve with the same epitope in different microenvironments.
Highlights
B-cell receptor associated protein 31 (BAP31) is a 28 kDa integral endoplasmic reticulum (ER) membrane protein and expressed ubiquitously [1,2,3]
The present study found that both monoclonal antibodies (MAbs) showed different binding patterns in flow cytometric analyses and quantitative binding studies, both recognized the same epitope on BAP31
We found that the two MAbs, 297-D4 and 144-A8, recognize a linear epitope on the C-terminal domain of BAP31 [14, 15]
Summary
B-cell receptor associated protein 31 (BAP31) is a 28 kDa integral endoplasmic reticulum (ER) membrane protein and expressed ubiquitously [1,2,3]. When cell surface expression of BAP31 was monitored on various cells by the two MAbs, 144-A8 exhibited an additional binding reactivity to A172, MDA-MB435 and NCI-H522 cells (S1 Fig and S1 Table), suggesting that the amino acid sequences of 144-A8 may be different from those of 297-D4, they recognize the same epitope.
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Disclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.